Nghiên cứu đột biến, mức độ biểu hiện gen EGFR và tình trạng METHYL hoá một số gen liên quan trên bệnh nhân ung thu biểu mô tuyến ở phổi tt tieng anh

MINISTRY OF EDUCATION

VIETNAM ACADEMY

AND TRAINING

OF SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY
----------------------------

NGUYEN NGOC QUANG

STUDY ON MUTATION, PROTEIN EXPRESSION
OF EGFR AND METHYLATION OF RELATED
GENES IN LUNG ADENOCARCINOMA PATIENTS
Major: Biotechnology
Code: 942 02 01

SUMMARY OF BIOLOGY DOCTORAL THESIS

Hanoi - 2020


The research was completed at: Graduate University Science and
Technology, Vietnam Academy of Science and Technology

Supervisor 1: Assoc. Prof. Chu Hoang Ha
Supervisor 2: PhD. MD. Nguyen Phi Hung

Reviewer 1: …

Reviewer 2: …
Reviewer 3: ….

The thesis will be defended in front of the Academy Thesis Evaluation
Council at graduate university science and technology, vietnam
academy of science and technology at …, date … month … year.

The thesis can be found at:
- Graduate university science and technology library
- Vietnam National library


OBJECTIVES
Lung cancer is the leading cause of cancer mortality in many
countries. The activating mutations in the tyrosine kinase (TK) domain
of epidermal growth factor receptor (EGFR) are considered as a
therapeutic target for lung cancer. Targeted treatment regimen by
tyrosine kinase inhibitors based on EGFR mutations have been widely
used and contributed to improve clinical management. On other hand,
EGFR protein expression is controlled by methylation level of it
promoter. Along with EGFR, aberrant epigenetic alterations of tumor
suppressor genes such as BRCA1, MGMT, MLH1, RASSF1A are
associated with the initiation and development of non-small cell lung
cancer. The EGFR mutation has been reported in lung cancer partients
in many recent studies. However, the molecular alterations of EGFR
and the effect of methylation of tumor suppressor genes on these
changes are not comprehensively investigated. Therefore, the goal of
this PhD research was aimed to investigate: "Study on mutations,
expression of EGFR and methylation of related genes in lung
adenocarcinoma patients".

Objectives
1. Analysis of mutation and protein expression of EGFR in patients
with lung adenocarcinoma.
2. Investigation the hypermethylation frequency of EGFR, BRCA1,
MGMT, MLH1, RASSF1A and correlation of the methylation between
these genes with mutations and protein expression of EGFR.
Dominant results
1. To collect medical records including lung adenocarcimoa and
benign specimens using lung screen.

1


2. To detect genetic and epigenetic alterations as well as protein
expression of EGFR and to investigate the association of molecular
abnormalities and clinicopathologic factors.
3. To determine the methylated promoter of tumor suppressor genes
such as BRCA1, MGMT, MLH1, and RASSF1A and evaluate the
relationship of their methylation with clinicopathologic parameters.
4.

To analyze the interaction between molecular abnormalities of

EGFR and hypermethylation of BRCA1, MGMT, MLH1, or RASSF1A.
CHAPTER 1. BACKGROUND
1.1. Lung cancer
1.1.1. Lung cancer facts
Lung cancer (LC) is the most common cancer in the world.
Vietnam is among the second highest rate of lung cancer in the
world, with the incidence in men is 25.5 - 41.5 /100 000 people and

in women is 7.3 - 13.6 /100 000 people.
1.1.2. Classification of lung cancer
Lung cancer is classified based on histopathological results
into two groups: on-small cell lung cancer (NSCLC) and small cell
lung cancer (SCLC). In which, adenocarcinoma (AD) is the most
common form of lung cancer
1.1.3. Stages of lung cancer
Currently,

the

most

commonly

used

lung

cancer

classification system is the TNM system (Tumor - Node Metastasis), Depending upon tumor size, the number of lymph nodes
metastasis and the degree of distant metastasis. According to this
classification system, the progression of lung cancer can be divided
into four stages from 0 to IV.

2


1.2. Adenocarcinoma lung cancer

1.2.1. Characteristics of adenocarcinoma lung cancer
Lung adenocarcinoma accounts for about 40% of lung
cancer. It usually starts in normal secretory cells such as mucus
secretions and more common in non-smokers, females and young
people.
1.2.2. The histopathological subtypes in lung adenocarcinoma
Tumors in adenocarcinoma lung cancer differentiate with one
or more developmental forms including several types: lepidic
predominant, acinar, papillary, micropapillary, solid …
1.3. EGFR alterations in lung cancer
1.3.1. EGFR gene structure and function
EGFR belongs to Epidermal Growth Factor Receptor
(EGFR) with 3 parts: Extracellular ligand binding, transmembrane
and tyrosine kinase activation domain. EGFR plays an important role
in the process of multiplication, apoptosis, cellular, invasion, repair
and interaction between cells.
1.3.2. EGFR mutation in lung cancer
Up to date, more than 40 EGFR mutations have been found
scattered on 4 exons from 18 - 21 in the kinase domain, which are
important in assessing the ability to treat lung cancer with TKIs.
1.3.3. EGFR expression in lung cancer
Promoter methylation is closely related to protein expression.
EGFR expression study facilitates the development of useful
biomarkers in clinical trials.
1.4. Methylation of DNA in lung cancer
1.4.1. Methylation of DNA

3

Methylation of DNA is the phenomenon of attaching -CH3 to
the 5'C position of nucleotides. Methylation of DNA plays roles in
normal cells such as gene expression, maintaining heterochromatin,
maintaining an inactive state of an X chromosome in female
mammals. DNA methylation is divided into two categories: hypomethylation and hyper-methylation.
1.4.2. Methylation of DNA in lung cancer
DNA methylation is a molecular marker in the early stage of
cancer, supporting the diagnosis, prognosis and treatment of lung
cancer. In particular, EGFR methylation is directly related to the
effectiveness of treatment with target drugs. In addition, the
methylation of tumor suppressor genes such as BRCA1, MGMG,
MLH1, RASSF1A i.e as been shown the role in predicting the
progression of lung cancer patients.
1.5. Targeted therapy in lung cancer
1.5.1. Targeted therapy in cancer
The targeted treatment in cancer is divided into 3 main
categories: Monoclonal antibodies, small molecule inhibitors and
immunotoxicity.
1.5.2. Targeted therapy in lung cancer
Currently, the FDA has approved EGFR monoclonal
antibodies including Cetuximab and Panitumumab; TKIs inhibitors
such as Erlotinib, Gefitinib, Osimertinib; TKK inhibitors of ALK
such as Crizotinib, Ceritinib for lung cancer treatment.
1.6. Methodological analysis of molecular alterations in lung
cancer
1.6.1. Methodological analysis of mutation in lung cancer

4

Gene mutations in lung cancer are detected through many
methods such as: DNA sequencing, Realtime PCR, DNA
hybridization…
1.6.2. Methodological analysis of DNA methylation in lung cancer
Three common methods are used to analyze DNA
methylation including: Immune precipitation; using methyl sensitive
enzyme and based on the bisulfite treated DNA.
1.7. Study on bio markers for lung cancer in Vietnam
In order to support the lung cancer targeted treatment in
Vietnam, serveral studies have been performed to determine the
EGFR mutation in NSCLC. For example, Vu A.H. et al., Nguyen M.
H. et al., Mai T. K. et al. studied on 332, 120 and 511 patients with
NSCLC and found that the EGFR mutation rate was 40.7, 35.7 and
40.1%, respectively. Furthermore, there were serveral thesises
focusing on EGFR mutation lung cancer. However, there is no
simultaneous evaluation of EGFR molecular changes including
genetic mutations, DNA methylation, and protein overexpression
along with methylation status of tumor suppressor genes such as
MGMT, MLH1, BRCA1, RASSF1A has been published. This can be
considered a new field of research for lung cancer in Vietnam.
CHAPTER 2. MATERIALS AND METHODS
2.1. Materials
139 samples of lung tumor, 5 adjacent lung cancer samples,
healthy human blood samples were provided by Hospital K. The use of
patient samples was approved by the ethicial committee of Hospital K.
The chemicals and kits used in the study were qualified for
molecular biology analysis materials.

5

2.2. Equipments
Specialized equipments for molecular biology analysis.
2.3. Methods
This thesis research was performed at the key gene technology
laboratory, Institute of Biotechnology and Molecular Biology
laboratory, Pathology – Molecular Biology Center, Vietnam National
Cancer Hospital. It has been carried out by cross-sectional description
method,

using

techniques

including:

Total

DNA

extraction,

determination of DNA concentration; total DNA bisulfite treatment;
PCR; MS-PCR; electrophoresis; identify EGFR mutations;
CHAPTER 3. RESULTS
3.1. Patient characteristics
In total of 139 patients in this study, the medium age of the
patient group was 57.4. Of which 94 patients are male and 45 patients
are female. There are 79 smoking patients, of which 76/79 cases were
male. Patients in the study was belong to three main histopathological

subtypes: Acinar adenocarcinoma (56.8%), Papillary adenocarcinoma
(15.8%) and Solid adenocarcinoma (24.5%). 104 samples were
collected from primary tumors and 35 samples from metastasis tumors.
Most patients were in stage II&IV (127/139) and there were only 12
cases in stage I&II.
3.2. EGFR molecular characteristics in lung adenocarcinomas
3.2.1. EGFR mutation and correlation with patient characteristics
EGFR mutation was detected in 35.3% (49/139) patients, with
12 different types. The deletion mutation at exon 19 and the
substitution mutation at exon 21 (L858R) accounted for 85.5%. In
addition, there were six cases carried two mutations simultaneously.

6


The substitution mutation G719X usually occured concurrently with
other mutations (4/5 cases) such as S768I, L861Q, L858R and 19
deletions. EGFR mutation was higher in younger patients, women and
non - smokers. At the same time, patients with solid lung
adenocarcinoma had a lower rate of mutation compared to the other
subtypes (Table 3.3).
Table 3.3. EGFR mutation and the correlation with clinicopathologic
parameters
EGFR mutation
Mutation

N
Age (57.4 ±10.8)
≤57.4
>57.4

Gender
Male
Female
Smoking status
Smoker
Non-smoker
Histological subtypes
Acinar
Papillary
Micropapillary
Solid
Mixed
Tumors
Primary
Metastasis
Stages
I & II
III & IV

p

Wildtype

139

49

90

67

72

30
19

37
53

94
45

23
26

71
19

79
60

20
29

59
31

79
22
3
34

1

32
7
2
7
1

47
15
1
27
0

104
35

36
13

68
22

12
127

6
43

6

84

0.023

<0.001

0.005

0.155
0.811
0.284
0.042
0.353
0.787

0.263

7


3.2.3. EGFR expression and correlation with patient characteristics
EGFR

protein

expression

was

assessed

by

immunohistochemistry. The results showed that 57 (41.0%) samples
were negative (IHC: 0, 1+) and 82 (59.0%) samples were positive
(IHC: 2+, 3+) with EGFR expression. There was no correlation
between EGFR expression and patient characteristics including age,
gender, smoking status, histologic subtype, metastasis status, or
pathologic stage (p>0.05) (Table 3.6).
Table 3.6. EGFR expression and correlation with
clinicopathologic parameters
EGFR overexpression
p
Negative

N
Age (57.4 ±10.8)
≤57.4
>57.4
Gender
Male
Female
Smoking status
Smoker
Non-smoker
Histological subtypes
Acinar
Papillary
Micropapillary
Solid

Mixed
Tumors
Primary
Metastasis
Stages
I & II
III & IV

Negative

139

57

82

67
72

29
28

38
44

94
45

35
22

59
23

79
60

31
26

48
34

79
22
3
34
1

27
9
2
18
1

52
13
1
16
0

104
35

39
18

64
18

12
127

4
32

8
95

0.599

0.191

0.627

0.060
0.992
0.361
0.104
0.229

0.203

0.572

8


3.3. Methylation of some genes involved in lung adenocarcinoma
3.3.1.

Methylation

of

EGFR

and

correlation with

patient

characteristics
Aberrant promoter methylation of EGFR was detected in 33
(23.7%) of a total of 139 lung adenocarcinomas. No significant
association between EGFR methylation and clinicopathologic variables
was observed.
3.3.2. Methylation of BRCA1 and correlation with patient
characteristics
Aberrant promoter methylation of BRCA1 was determined in

41 (29.5%) of a total of 139 lung adenocarcinomas. No significant
association between BRCA1 methylation and patient characteristics
was indicated.
3.3.3. Methylation of MGMT and correlation with patient
characteristics
Aberrant promoter methylation of MGMT was detected in 46
(33.1%) tumors of a total of 139 lung adenocarcinomas. Furthermore,
aberrant MGMT methylations was associated with metastasis status
(p<0.05), but not with other clinicopathologic features.
3.3.4. Methylation of MLH1 and correlation with patient
characteristics
Our results showed that aberrant promoter methylation of
MLH1 was detected in 28/139 (20.1%) of a total of 139 lung
adenocarcinomas.

No

significant

association

between

MLH1

methylation and patient characteristics was observed.
3.3.5. Methylation of RASSF1A and correlation with patient
characteristics

9

Aberrant promoter methylation of RASSF1A was detected in
41 (29.5%) tumors of a total of 139 lung adenocarcinomas. The
statistically significant association between RASSF1A methylation and
smoking status was observed. Prevalence of methylation in smokers
was higher than that in non-smokers (p<0.05). Furthermore, aberrant
RASSF1A methylation were also associated with metastasis status
(p<0.05) (Table 3.11).
Table 3.11. Methylation of RASSF1A and correlation with patient
characteristics

N
Age (57.4 ±10.8)
≤57.4
>57.4
Gender
Male
Female
Smoking status
Smoker
Non-smoker
Histological subtypes
Acinar
Papillary
Micropapillary
Solid
Mixed
Tumors
Primary

Metastasis
Stages
I & II
III & IV

Methyl RASSF1A
M
U
41

139

p
98

67
72

20
21

47
51

0.930

94
45

29

12

65
33

0.613

79
60

29
12

50
48

0.032

79
22
3
34
1

22
7
1
11
0

57
15
2
23
1

0.625
0.795
0.883
0.674
0.521

104
35

26
15

78
20

0.045

12
127

4
37

8

90

0.760

3.4. Correlation between mutation and expression of EGFR with
methylation of related genes in lung adenocarcinoma

10


3.4.1. Correlation between mutation, protein expression and DNA
methylation of EGFR
The mutation status exhibited no significant association with
promoter methylation and protein overexpression of EGFR (p>0.05)
but statistically correlated between EGFR methylation status and its
protein expression was observed (p<0.05) (Table 3.12).
Table 3.12. Correlation between mutation, protein expression and DNA
methylation of EGFR
EGFR methylaion

EGFR expression
EGFR mutation

+
+
-

EGFR mutaion

M

33

U
106

p

+
49

90

p

21
12
15
18

36
70
34
72

0.002

20
29

37

53

0.973

0.160

3.4.2. Correlation between mutation and expression of EGFR with
methylation of BRCA1, MGMT, MLH1 and RASSF1A
The distribution of EGFR mutations and BRCA1, MGMT, or
RASSF1A

methylation

were

significantly

exclusive

in

lung

adenocarcinomas. Furthermore, EGFR mutation inversely correlated
with the methylation of at least one of the four genes, at least two of
four genes or three genes (Table 15).
On other hand, EGFR expression did not correlate with
MGMT, MLH1, or RASSF1A methylation. However, BRCA1
methylation was indicated to correlate with EGFR expression
3.4.3. Correlation of methylation between genes related to lung

adenocarcinoma
In more detail, we analyzed the relationship between
methylation of each set of two out of five genes. The results showed

11


that RASSF1A methylation was correlated with BRCA1 and MLH1
(Table 17).
Table 3.15. Correlation of EGFR mutation and BRCA1, MGMT, MLH1, and
RASSF1A methylation
EGFR Mutation
N

Mutation

Wildtype

139

49

90

M

41

9

32

U

98

40

58

M

46

11

35

U

93

38

55

M

28

8

20

U

111

41

70

M

41

7

34

U

98

42

56

M

90

26

64

U

49

23

26

M

41

6

35

U

98

43

55

M

17

1

16

U

122

48

74

M

5

0

5

U

134

49

85

Methylation
BRCA1
MGMT
MLH1
RASSF1A
1 in 4
2 in 4
3 in 4
4 in 4

p
0.034
0.049
0.408
0.004
0.041
0.001
0.004
0.106

CHAPTER 4. DISCUSSION
4.1. EGFR molecular characteristics in patients with lung
adenocarcinoma
4.1.1. EGFR mutation at Tyrosine Kinase Domain region
Analysis of 139 patients, we found that 35.3% of samples
occurred EGFR mutations, It is similar to previous reports in some

12

Asian countries such as Japan 32%, South Korea 36.4%; lower than
some other countries in the region such as Thailand, 57.4%, Taiwan is
55% and higher than the America and Europe (10 - 15%). EGFR
mutation occurs with high frequency in patients with lung
adenocarcinoma at National Cancer Hospital and more often in young
people, women and non-smokers. The solid adenocarcinoma has a
lower rate of mutation than other adenocarcinoma types (Table 3.3).
Midha A. et al. (2015) Summarized and analyzed 139 studies of EGFR
mutations around the world

and indicated that although there are

differences in mutation rates among groups of people, geographic
regions, the rate of EGFR mutations is always higher than in women
and non-smoker patients.
Table 3.17. Correlation of tumor suppressor genes methylation
EGFR

BRCA1
MGMT
RASSF1
MLH1

MLH1

RASSF1A

MGMT

M

U

p

M

U

p

M

U

p

M

U

p

M

11

30

0.580

10

31

0.419

21

20

0.001

15

26

0.571

U

22

76

18

80

20

78

31

67

M

13

33

13

33

17

29

U

20

73

15

78

24

69

M

10

31

17

24

U

23

75

11

87

M

10

18

U

23

88

0.378
0.907

0.093

0.175

0.001

0.096

4.1.2. Overexpression of EGFR protein
Evaluation of EGFR expression shows that 59.0% (82/139) of
tumor samples were positive. EGFR expression in this study is in the
same level with those reported from previous studies by Cappuzzo F.
et al. (2005) and Hirsch F.R. et al. (2008). Furthermore, we found that
EGFR expression did not correlate with clinicopathologic variables
13


(Table 3.6); which was also observed in the study of Cappuzzo F. et al.

(2005) and Liang Z. (2010). In addition, we found that overexpression
of EGFR (3+) in men was higher than in women. However, the
mechanism of this phenomenon has not been clarified. Moreover, the
level of EGFR expression in metastatic tumors tended to increase
compared to those in primary tumors. Thus, it can be assumed that the
higher degree of malignancy in metastasis tumors is the result of EGFR
overexpression.
4.1.3. Methylation of EGFR promoter region
The frequency of methylated EGFR in this study was 23.7%,
that was lower than that of Li J. et al. (2015) (36.8%) and Pan Z.Y. et
al. (2015) (35.7%). We did not find a correlation between EGFR
methylation with patient characteristics such as age, gender, smoking
status, histological subtypes, stage of disease and tumor status; The
similar results were also indicated the previous publications. However,
Li J. et al. (2015) found that patients in stage III have lower levels of
methylated EGFR than stages I and II.
4.1.4. Correlation between EGFR molecular characteristics
EGFR mutations lead to changes in amino acid sequence as
well as protein activity thereby affecting cell growth and development.
However, the level of protein expression is controlled by many
pathways in the cell including promoter methylation. Therefore,
studying the correlation of mutations, methylation and protein
expression will contribute to elucidating the mechanism of cancer
formation and development.
Studied on the correlation between gene mutation and
methylation as well as EGFR expression showed very different results.
Analysis of 139 patients, we found that there is no correlation between

14

mutation with methylation and EGFR protein expression. However,
there was a significant association between EGFR promoter
methylation and EGFR protein expression (Table 3.12). Cappuzzo F. et
al. (2005) did not find the correlation between EGFR expression and
EGFR mutation; In contrast, Liang Z. (2010) found a high level of
EGFR expression in patients with EGFR mutations. The correlation of
mutation, methylation and expression EGFR may be different between
the research groups due to differences in ethicity, age, gender and stage
of disease. Protein expression levels can be used in responding
evaluation as well as prognosis in cancer treatment. Li et al. (2013)
showed an increase in EGFR expression and programmed death in
methylated EGFR cell lines cultured in media added 5-aza-CdR and
TKIs. Therefore, regulation EGFR expression levels by methylated
DNA which combines with TKIs targeted therapy could be a new
direction not only in research but also in the treatment of lung cancer.
4.2. Methylation of tumor suppressor genes BRCA1, MGMT,
MLH1 and RASSF1A in patients with lung adenocarcinoma
4.2.1. Aberrant promoter methylations of BRCA1
Methylated BRCA1 was commonly studied in breast cancer but
had little attention in lung cancer. The hypermethylation of BRCA1 can
be considered a tumorigenesis pathway in addition to the EGFR or
KRAS mutation. The rate of BRCA1 methylation in various studies
ranges from 4 - 54%. The frequency of methylated BRCA1 in this
study was 29.5% which was higher than in American patients (4%) and
lower than in Chinese (30 - 54%). This results could be explained by
the differences in ethicity and geography of patient populations. In
Asian populations, BRCA1 methylation tends to occur more commonly
than Western patients.

15


In this study, there was no correlation between methyl BRCA1
and patient characteristics. In previous studies, they were not although
fully studying the features, but most of them showed no correlation
between clinicopathologic variables and methylation BRCA1. Gao W.
et al. (2016) indicated that BRCA1 methylation tended to increase in
stage II and III than stage I; in patients with lymph node metastasis
compared with patients without lymph node metastasis. From these
results can be assumed that methylated BRCA1 occurs in parallel with
the progression of the disease, the more the disease progresses the
greater the level of methylation. In this study, we also found an
increase in the methylation status of BRCA1 with disease progression
but it is not significant. Therefore, methylation of BRCA1 can be
considered as one of the causes leading to the tumorigenesis and
development of lung adenocarcinoma and needs to be studied in detail.
4.2.2. Aberrant promoter methylations of MGMT
MGM methylation has been observed in many cancers
including lung cancer. The percentage of MGMT methylation was in
various studies from 8 to 50%. The frequency of methylated MGMT in
this study was 33.1%; It was higher than the reports of Feng Q. et al.
(2008) (8%), Kim et al. (2005) (17%); and similar to other researches
which were performed on Chinese patients (30-50%). MGMT
methylation inhibits protein expression and reduces the MGMT DNA
repair function leading to increase the mutation establishment. The
results of correlation analysis showed that MGMT methylation is not
associated with the patient characteristics in the study excepted
metastasis status. Reports on Korean and Chinese patients have
showed similar results. In this study, we found an increase in the

MGMT methylation in metastatic tumors. Although the mechanism for

16


promoting methylated MGMT in metastatic cells in lung cancer has not
been elucidated, it can be seen that MGMT methylation may contribute
to accelerating tumor metastases and disease progression. MGMT
methylation could be considered as one of the standard markers
supporting the prognosis and treatment of lung cancer.
4.2.3. Aberrant promoter methylations of MLH1
Study on MLH1 methylation is popular in lung cancer, but the
rate of methylation varies widely among the studies. The previously
published MLH1methylation frequency ranges from 0 to 58%. The
ratio of methylataed MLH1 in our research was 20.1% which is higher
than a report of Tang M. et al. (2006) (0%); and lower than publishes
in Asian patients (30-50%). In addition, Seng T.J. et al. (2008)
compared survival rates of methylated and unmethylated MLH1
patients, and suggested that patients with the MLH1 methylation had a
poor prognosis and lower survival time than the unmethylated group.
MLH1 methylation could be one of the standard markers in the
prognostic assessment for lung cancer patients along with other
markers.
MLH1 methylation studies on different patient groups have
been performed and showed no correlation between MLH1 methylation
with patient features such as age, gender, smoking status. The reports
often did not go into the different subtypes of adenocarcinoma, but
only focus on patients with adenocarcinoma. However, the status of
methylated MLH1 at different stages of the disease showed
inconsistencies between the studies. In the publication of Seng T.J. et

al. (2008), the author found that the rate of methylation MLH1 in
patients with stage II was higher than in stage I (40/86 compared to
46/153). In contrast, Wang Y.C. et al. (2003) indicated that the

17


difference in MLH1 methylation level in the group of patients with
advanced stage and early stage patients (stage III and IV compared
with stages I and II) was not statistically significant. In this study, we
also did not observe differences in the level of methylation MLH1 at
different stages of the disease (patients in the study were
predominantly in late stages) as well as at the tumor status (primary or
metastatic tumors). With the function of DNA mismatches repair in
replication process, it is possible to hypothesize that the MLH1
methylation occurs mainly during the invasive stage of the tumor when
cancer progress from stage I to stage II. In this process, the DNA
mismatch repair function of the cell is impaired and the secondary
mutations are generated before entering the metastatic stage.
4.2.4. Aberrant promoter methylations of RASSF1A
RASSF1A is involved in regulating many important processes
in the cell including the cell cycle as well as the programmed death
process. Methylation of RASSF1A is crucial to inhibiting expression of
this protein. Therefore, RASSF1A methylation has become a standard
marker in the diagnosis and treatment of lung cancer. The rate of
methylated RASSF1A in the previous studies varies from 20 to 80%.
The frequency of RASSF1A methylation in our study was 29.5% (Table
2.17). As reported by de Fraipont F. et al. (2012) on overall survival
and progression-free survival of two groups of patients after
chemotherapy. The results showed that the methylated RASSF1A group

had shorter survival time than the unmethylated group (progressionfree survival 16.7 versus 61.2 months and overall survival 32.9
compared to 84 months). It can be seen that determination of RASSF1A
methylation status should be considered as one of the prognostic
factors for lung cancer patients.

18


Methylation of RASSF1A has been associated to smoking
habit, and the correlation between RASSF1A methylation and smoking
status of patients has also been published in several studies, such as
Yanagawa N. et al. (2011) and Lee S.M. et al. (2011). Therefore,
smoking can be the cause of methylated RASSF1A in patients with lung
cancer. Besides the EGFR mutation, methylation of RASSF1A may be
an important mechanism in the carcinogenesis and development of
lung cancer. Previously, RASSF1A has also been showed to inhibit the
invasion and metastasis of lung cancer cell lines by prohibiting YAP
(Yes-associated protein) protein. In our study, the level of RASSF1A
methylation in metastatic tumors was higher than in primary tumors.
The results proved that methylation of RASSF1A is not only involved
in tumorigenesis but also plays an important role in tumor metastasis in
lung cancer. Therefore, the identification of RASSF1A methylation
status is becoming increasingly necessary for patients, contributing to
the diagnosis as well as prognosis.
4.3. Correlation of EGFR alterations with aberrant promoter
methylations of BRCA1, MGMT, MLH1, and RASSF1A in lung
adenocarcinoma
4.3.1. Correlation of EGFR mutation with methylation of tumor
suppressor genes
Gene mutations and DNA methylation are the two main

pathways for cancer generation and development. The interaction of
these two pathways has been found in many types of cancer. In this
study, we analyzed the interaction between EGFR mutation and
methylation of four tumor suppressor genes typical of lung
adenocarcinoma. Results showed that EGFR mutation and methylation
of tumor suppressor genes tended to occur in opposite directions and

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mutually exclusive (Table 3.1). The inversed correlation between
EGFR mutation and RASSF1A methylation has been mentioned in a
number of previous studies. Yanagawa N. et al. (2011) and Hoque
M.O. et al. (2010) showed an inversed association simultaneously
between RASSF1A methylation with smoking status and EGFR
mutation. This study has confirmed the mutual exclusion of these two
phenomena. At the same time, with the above analysis, EGFR mutation
occurs in non-smokers, whereas methylation of RASSF1A is common
in smokers. Therefore, it can be hypothesized that in non-smokers,
adenocarcinoma lung cancer may be established by EGFR mutation. In
contrast, lung adenocarcinoma in smokers are formed via the
methylation of RASSF1A.
In addition, in the study, we also showed the inversed
correlation between EGFR mutation and methylation BRCA1 and
MGMT (Table 3.15). The study of methylation of BRCA1 and MGMT
has received limited attention in lung cancer. However, our results
showed that BRCA1 methylation and MGMT methylation were
common in lung cancer, and occured primarily in patients without
EGFR mutations. RASSF1A methylation not only is considered one of
the pathways for the formation of lung adenocarcinoma, the

methylated BRCA1 and MGMT can also be an important step in tumor
formation and development in patients without EGFR mutations. This
theory was further strengthened by the results of analyzing the
correlation of methyl and the four genes BRCA1, MGMT, MLH1 and
RASSF1A with EGFR mutations (Table 3.15). The inversed correlation
of EGFR mutation with random methylation of an individual gene,
simultaneous 2, 3 or 4 genes found a clearer observation of genetic and
epigenetic changes in lung adenocarcinoma. Our results have also

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showed that gene mutation and DNA methylation of tumor suppressor
genes are two pathways leading to the formation and development of
lung cancer. Therefore, it can be considered that DNA methylation
needs to be researched and tested as a target for cancer treatment.
4.3.2. Correlation of EGFR expression with methylation of tumor
suppressor genes
In this study, we found that EGFR expression increased in
patients without methylated BRCA1. Although the mechanism of
correlation between BRCA1 methylation and EGFR protein expression
has not been clarified, it can be seen that methylaed BRCA1 is involved
in inhibiting EGFR expression. However, there were a few studies on
lung cancer have been conducted to clarify the mechanism of this
phenomenon. Correlation between methylation of BRCA1 and EGFR
expression was investigated in ovarian cancer, but the results were
opposite. Da Li et al. (2013) suggested that BRCA1 methylation may
promote EGFR expression in ovarian cancer cell lines. The difference
of two researches may help to explain the role of different genes in
controlling cellular processes in cancer.

The random methylation of one in four genes also showed a
reversed correlation between the methylation of tumor suppressor
genes and EGFR expression. Tumor suppressor gene methylation
showed more effectively in patient groups which differs in disease
stage and tumor status. The studies of Gao W. et al. (2016), Wang Y.C.
et al. (2003) and Lee S.M. et al. (2011) showed higher levels of
methylated BRCA1, MLH1 and RASSF1A in late-stage patients;
meanwhile Li J. et al. (2015) indicated a decrease in EGFR expression
according to disease progression. In this study, we found that MGMT
methylation, and RASSF1A in metastatic tumors was higher than

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primary tumors; in contrast, EGFR expression was lower in metastatic
tumors than in primary tumors. From of our findings, tumor suppressor
gene methylation and EGFR expression are two opposing trends in
lung carcinoma. It can be hypothesized that methylation of tumor
suppressor genes tends to inhibit the cell growth factor receptors.
Specifically, in this study, we found that there was a decrease in EGFR
protein expression, an important receptor in the development of both
normal cells and lung adenocarcinomas.
4.3.3.

Simultaneous

methylation

of

genes

related

to

lung

adenocarcinomas
EGFR methylation leads to inhibition of protein expression
and can be considered as a defensive mechanism to limit the growth of
cancer cells. Meanwhile, methylation of tumor suppressor genes
promotes cancer development. However, the results of this study
showed that the EGFR methylation occurred independently with the
methylation of BRCA1, MGMT, MLH1 and RASSF1A (Table 3.17).
The methylation of tumor suppressor genes in previous publications on
lung cancer has related to poor prognosis for patients and inversed
correlation with EGFR mutations, but it is not statistically significant
with methylated EGFR. It can be assumed that BRCA1, MGMT, MLH1
and RASSF1A methylation and EGFR methylation are two independent
processes in lung adenocarcinoma. Methylation of tumor suppressor
genes contributes to the development of disease. In contrast, EGFR
methylation plays a role in limiting tumor growth in lung cancer.
Genetic and epigenetic alterations of tumor suppressor genes
and oncogenes are important mechanisms in the formation and
progression of many types of cancer including lung cancer.
Understanding of the extent of abnormal DNA methylation in tumors

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contributes to elucidate the events that occur in cancer and also
provides effective treatment pathways. In this study, we analyzed
methylation of BRCA1, MGMT, MLH1 and RASSF1A. There have
been reports of simultaneous methylation in many genes in lung
cancer. However, there is very little DNA methylation research on the
correlation of tumor genes. Seng T.J et al. (2008) have showed a
positive correlation between methylated RASSF1A and methyl MLH1.
Results of this study also confirmed that patients with methylation
individually or simultaneously RASSF1A with MLH1 have a shorter
survival time than unmethylated patients. This suggests that tumor
suppressor genes are not only working alone but also co-working with
other ones. In this study, we found an additional correlation between
RASSF1A and BRCA1 methylation (Table 3.17). According to Gao W.
et al. (2016), methylated RASSF1A and BRCA1 have worse prognosis
than unmethylated cases. However, the author did not provide a
correlation between methylation of RASSF1A and BRCA1. Therefore,
more methylation of tumor suppressor genes leads to higher the
malignancy of the tumor and worse the prognosis for patients.
CONCLUSION
1. The rate of EGFR mutation and EGFR overexpression in this study
was 35.3 and 59.0 percent, respectively. The frequency of EGFR
mutations was higher in younger, women and non-smoking patients. In
addition, EGFR overexpression was higher in men than in women.
2. The methylation rate of EGFR, BRCA1, MGMT, MLH1 and
RASSF1A genes related to lung cancer was 23.7, 29.5, 33.1,20.1 and
29.5 percent, respectively. MGMT and RASSF1A methylation had a
higher incidence in metastatic tumors; RASSF1A methylation was
higher in non-smokers and occurred simultaneously with BRCA1 and

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